Recombinant SEMG1214-42was biotinylated as described previously [23]

Recombinant SEMG1214-42was biotinylated as described previously [23]. == In Vitro Site-Directed Mutagenesis == Site-directed mutagenesis of wild-type SEMG1214-42of residue Cys239 to glycine (C239G-SEMG1), aspartic acid (C239D-SEMG1), histidine (C239H-SEMG1), serine (C239S-SEMG1), or arginine (C239R-SEMG1) was performed using the Gene Tailor site-directed mutagenesis system (Invitrogen), according to the manufacturers instructions. reduced capacity to interact to EPPIN and to inhibit human sperm motility in vitro. In conclusion, our results indicate that EPPIN and SEMG1 rapidly co-evolved in primates due to their critical role in the modulation of sperm motility in the semen coagulum, providing unique insights into the molecular co-evolution of sperm surface interacting proteins. == Introduction == In primates, semen is Eptifibatide a complex biological fluid containing spermatozoa bathed by the seminal plasma, which nurtures spermatozoa while providing the appropriate conditions for the last steps of post-testicular sperm maturation, and protecting them from pathogenic threats during their journey towards fertilization in the female reproductive tract [1]. The seminal vesicle-secreted protein semenogelin I (SEMG1) is the major component Eptifibatide of the Sox2 seminal plasma [2]. Immediately after ejaculation, human semen undergoes a coagulation process forming a gelatinous mass that contains SEMG1 as its structural element [3]. SEMG1 is found in the semen coagulum and on the surface of spermatozoa bound to EPPIN, a member of the whey-acidic protein (WAP)-type four-disulfide core (WFDC) Eptifibatide family [4,5]. After ejaculation, the hydrolysis of SEMG1 by activated prostate-specific antigen (PSA), a serine protease, results in the liquefaction of the semen coagulum allowing spermatozoa to acquire progressive motility [2,6]. The physiological significance of the SEMG1/EPPIN interaction on the surface of spermatozoa is its capacity to modulate sperm progressive motility upon ejaculation in a finely time-regulated manner [5,7]. In parallel, EPPIN inhibits the digestion of SEMG1 by PSA, which results in the modulation of semen liquefaction, further prolonging the inhibitory effects of SEMG1 on sperm motility [8]. This effect is thought to be important for reproduction because it allows spermatozoa to achieve their full fertilizing capacity at the appropriate moment. Men whose semen coagulum fails to liquefy spontaneously have spermatozoa with poor motility and are infertile [9]. Additionally, EPPIN and some SEMG1-proteolytic peptides derived by PSA cleavage have strong antibacterial activity in vitro and may protect the integrity of spermatozoa against microorganisms present in the vaginal environment [10-13]. The critical roles of SEMG1 and EPPIN in reproduction are highlighted by the fact that male monkeys Eptifibatide immunized with human recombinant EPPIN that developed high titers of anti-EPPIN antibodies lacked semen coagulum upon ejaculation and became reversibly infertile [14]. Subsequent studies demonstrated that anti-EPPIN antibodies isolated from immunized monkeys blocked the SEMG1 binding site on Eptifibatide EPPINs C-terminal region and mimicked SEMG1 binding by inhibiting progressive motility of human spermatozoa [15,16]. Because of these observations, the SEMG1 binding interface on EPPIN has been acknowledged as a potential target for male contraception [5]. The WFDC locus on human chromosome 20q13, containing theSEMG1(in the centromeric cluster) andEPPIN(in the telomeric cluster) genes [17,18], has undergone strong adaptive pressure [19] and theSEMG1gene in particular has undergone rapid adaptive evolution [18-21], suggesting positive selection driven by their functions in natural immunity and reproductive success [19]. Genes for serine protease inhibitors within the WFDC locus may have been progenitors to theSEMGgenes [17,22]. In previous studies on the interaction of SEMG1 with EPPIN on the sperm surface, we demonstrated that EPPINs Cys102, Tyr107, and Phe117 were necessary for SEMG1 binding [23] and that SEMG1s Cys239.